The Protective Effect Of Uric Acid Against6-OHDA-induced Injury In SH-SH5Y Cells Was Via Nrf2Signaling Pathway

PART ⅠProtective effect of uric acid on6-OHDA-induced injury inSH-SY5Y cells was associated with increased glutathione synthesisObjective: To investigatethe effect of uricacid (UA)on6-hydroxydopamine(6-OHDA)-induced injury in SH-SY5Y cells.Methods: SH-SY5Y cells were used to produce cell model of Parkinson’s disease andwere randomly divided into four groups and subjected to the following treatment: control,6-OHDA(50μmol/L), UA(200μmol/L) and6-OHDA (50μmol/L)+UA(200μmol/L). Cellswere treated with200μmol/LUA for0.5h prior to50μmol/L6-OHDA for6h,12h,14h,18h,24h, then cell viability was assessed by MTT assay; morphological changes were assessedby inverted microscope after12h treatment; glutathione content was assessed after12htreatment; γ-glutamylcysteine ligase catalytic subunit (γ-GCLC) and γ-glutamyl cysteineligase regulatory subunit (γ-GCLM) of protein expression and gene levels were assessedafter12h treatment by Western blot and RT-PCR methods.Results: Compared to50μmol/L6-OHDA group alone, pretreatment with200μmol/Luric acid for0.5h, cell viability of treatment for12h,14h,18h,24h on SH-SY5Y cellswas significantly improved (78.10±2.36%vs57.44±12.02%,80.52±3.16%vs50.15±2.32%,70.82±1.81%vs39.22±0.78%,42.84±4.73%vs27.99±4.23%, P <0.01, P<0.001, P <0.001, P <0.001); morphological changes were significantly alleviated after12h treatment; glutathione content after12h treatment was significantly increased (9.51±0.37μmol/Lvs3.88±0.78μmol/L, P <0.01);200μmol/L uric acid+50μmol/L6-OHDAgroup compared with50μmol/L6-OHDA group, γ-GCLC protein expression level wassignificantly increased (139.85±11.62%vs49.20±7.09%, P <0.01);. γ-GCLM proteinexpression level was significantly decreased (95.36±18.98%vs160.05±8.59%，P<0.05). γ-GCLC mRNA level was significantly increased (110.99±14.41%vs49.19±6.87%，P<0.001);. γ-GCLM mRNA level was significantly decreased (102.70±10.43%vs190.05±8.59%, P <0.05).Conclusions: Protective effect of uric acid on6-OHDA-induced SH-SY5Y cell injurywas associated with increased glutathione synthesis. PARTⅡRegulation of uric acid on the Nrf2signaling pathway.Objective: To investigate molecular mechanisms of increased glutathione contentinduced by uric acid.Methods: SH-SY5Y cells were randomly divided into four groups and subjected tothe following treatment: control,6-OHDA (50μmol/L), UA(200μmol/L),6-OHDA(50μmol/L)+UA(200μmol/L).Cells were pretreated with uric acid for0.5h, thenadministration with6-OHDA for6h, Nrf2distribution was observed by confocal imagingmethod and Nrf2cytoplasmic and nuclear protein extracts were determined by western blot.After transfection with Nrf2-siRNA, glutathione content and cell viability were determinedwith colorimetric method in different groups in Nrf2-siRNAgroup.Results: Compared with the control group, after50μmol/L6-OHDA tretatment onSH-SY5Y cells for6h, Nrf2distribution was no significantly nuclear translocation andcytoplasmic and nuclear protein extracts were not significantly changed; Nrf2wastranslocated from cytoplasm to nucleus after200μmol/L uric acid treatment for6hobserved by confocal imaging and Nrf2nuclear protein extracts were significantlyincreased (196.33±35.21%vs93.11±29.25%, P <0.05) compared with50μmol/L6-OHDA group alone; Nrf2nuclear protein extracts were significantly increased (231.36±55.19%vs100.00±33.01%, P <0.01) in200μmol/L uric acid group compared with controlgroup. In Nrf2-siRNA group,200μmol/L uric acid+50μmol/L6-OHDA group comparedwith50μmol/L6-OHDA group, glutathione content was not changed （4.16±0.12μmol/Lvs3.64±0.50μmol/L, P>0.05）；cell viability was not significantly increased（53.43± 2.21%vs51.37±1.69%, P>0.05）.Conclusion Protective effect of uric acid on6-OHDA-induced SH-SY5Y cell injurywas associated with Nrf2signaling pathway.